SUMMARY

Few studies have examined the adaptive significance of reversible
acclimation responses. The aerobic performance and mating behaviour of the
sexually coercive male eastern mosquito fish (Gambusia holbrooki)
offers an excellent model system for testing the benefits of reversible
acclimation responses to mating success. We exposed male mosquito fish to
normoxic or hypoxic conditions for 4 weeks and tested their maximum sustained
swimming performance and their ability to obtain coercive matings under both
normoxic and hypoxic conditions. We predicted that hypoxia-acclimated males
would possess greater swimming and mating performance in hypoxic conditions
than normoxic-acclimated males, and vice versa when tested in
normoxia. Supporting our predictions, we found the sustained swimming
performance of male mosquito fish was greater in a hypoxic environment
following long-term exposure to low partial pressures of oxygen. However, the
benefits of acclimation responses to mating performance were dependent on
whether they were tested in the presence or absence of male-male competition.
In a non-competitive environment, male mosquito fish acclimated to hypoxic
conditions spent a greater amount of time following females and obtained more
copulations than normoxic-acclimated males when tested in low partial
pressures of oxygen. When males were competed against each other for
copulations, we found no influence of long-term exposure to different partial
pressures of oxygen on mating behaviour. Thus, despite improvements in the
aerobic capacity of male mosquito fish following long-term acclimation to
hypoxic conditions, these benefits did not always manifest themselves in
improved mating performance. This study represents one of the first
experimental tests of the benefits of reversible acclimation responses, and
indicates that the ecological significance of physiological plasticity may be
more complicated than previously imagined.

Introduction

Many freshwater environments have extreme temporal and/or spatial variation
in the partial pressure of dissolved oxygen. The effective uptake of oxygen
from aquatic environments is critical to the survival of all fishes and
influences changes in their physiology, behaviour and distribution. Fish
possess a range of rapid behavioural and physiological responses that can
minimise the costs associated with decreases in the partial pressure of oxygen
by allowing either more effective uptake of oxygen or decreasing their
requirements. These include increases in the opercular beat rate
(Maxime et al., 2000), changes
in plasma pH causing a left shift in the haemoglobin-oxygen binding affinity
(Nikinmaa and Salama, 1969),
aquatic surface respiration (ASR) (Hughs,
1973; Kramer and Mehegan,
1981; Burggren,
1982; Kramer and McClure,
1982; Chapman et al.,
1995), behavioural hypothermia
(Jensen et al., 1993) and
microhabitat selection and movement patterns
(Wannamaker and Rice,
2000).

Long-term decreases in the partial pressure of oxygen can also elicit a
range of other physiological responses in fish that can allow more effective
uptake of oxygen under hypoxic conditions. For example, haematocrit,
haemoglobin and erythrocyte concentrations increase in some species after
several weeks of exposure to hypoxic conditions
(Frey et al., 1998;
Timmerman and Chapman, 2004b;
Jensen et al., 1993), whereas
the oxygen binding affinity can be modified in others
(Love and Rees, 2002). In
addition, increases in gill surface area have been reported in the sailfin
molly (Poecilia latipinna) acclimated to hypoxia
(Timmerman and Chapman,
2004a). However, despite the diversity of studies demonstrating
the physiological mechanisms underlying improved uptake of oxygen under
hypoxic conditions, few studies have addressed the ecological or fitness
consequences of short- or long-term changes in the partial pressure of oxygen
in the aquatic environment (Kramer and
Mehegan, 1981; Hale et al.,
2003). This is surprising given the obvious importance of
environmental oxygen availability on the evolution of freshwater biota.

Studying the ecological or fitness consequences of physiological responses
is often problematic because of the difficulties associated with linking
physiological traits with their direct reproductive or survival benefits
(Wilson and Johnston, 2004).
Most studies attempting to relate physiological traits to their ecological
significance use measures of locomotor performance as a proxy of fitness,
because of its presumed connection with escape from predators or prey-capture.
However, it is often not clear whether variation in locomotor performance
actually influences survival or reproductive success in the wild
(Walker et al., 2005). Linking
physiological studies with reproductive behaviour offers an alternative
opportunity for examining the direct implications of variation in
physiological function with reproductive performance. Behavioural ecologists
have already successfully utilised the mating behaviour of organisms to
examine the ecological and reproductive consequences of variation in both
morphological and behavioural attributes
(Condon and Wilson, 2006).

The effect of environmental oxygen on the mating behaviour of male eastern
mosquito fish offers an excellent opportunity for examining questions related
to the fitness consequences of variation in physiological traits. The mosquito
fish is a viviparous poeciliid fish native to the south-eastern USA and is an
introduced pest species across much of eastern Australia. Mosquito fish are
ideal for studies of acclimation as they inhabit a wide range of freshwater
environments from swampy hypoxic conditions to well-oxygenated streams.
Mosquito fish have been used extensively as a model system in studies of
sexual selection owing to the naturally high sexual activity of the males and
their coercive mating strategy (Bisazza et
al., 2001). Males never display to females, females never
cooperate and copulations are only achieved via a forced strategy.
Males approach the females with stealth and rapidly insert their gonopodium
into the female gonadopore to release their sperm. As the maintenance of these
reproductive behaviours (up to 90% of their total activity) is likely to be
dependent on their aerobic capabilities
(Wilson, 2005), the available
oxygen in the aquatic environment may be an important determinant of mating
performance. The ability of eastern mosquito fish to respond to long-term
changes in the partial pressure of oxygen in their aquatic environment offers
an excellent system to test the benefits of physiological acclimation.

We tested the ability of male mosquito fish to modify their reproductive
and locomotor performance to compensate for long-term changes in the partial
pressure of oxygen, allowing us to test the adaptive benefits of reversible
acclimation. We exposed male mosquito fish to either normoxic or hypoxic
conditions for a period of at least 6 weeks and tested their maximum sustained
swimming performance and their ability to obtain coercive matings under both
normoxic and hypoxic conditions. We predicted that hypoxia-acclimated males
would possess a greater sustained swimming performance and obtain more
coercive copulations in hypoxic conditions than normoxic-acclimated males, and
vice versa when tested in normoxia. We also examined the total sperm
stores of the different treatment groups and predicted that hypoxic-acclimated
males would have smaller ejaculate sizes due to the greater energetic costs of
inhabiting a hypoxic environment.

Methods and materials

Eastern mosquito fish Gambusia holbrooki Girard were collected
from 18 Mile Swamp on North Stradbroke Island, Queensland. Fish were returned
to the University of Queensland and maintained in single-sexed groups in 60 l
aquaria and fed daily on flaked fish food. After at least 2 weeks in the
laboratory, approximately 60 males and 60 females were separated into two
treatment groups; hypoxia acclimation and normoxia acclimation (14 h:10 h
light:dark). For all experimental tanks, hypoxic conditions were maintained by
bubbling commercially purchased N2 through a single air stone and
decreasing any surface air movement by fitting tanks with glass lids. Partial
pressure of oxygen for the hypoxic treatment was maintained between 3.1-4.0
kPa (approx. 15-20% saturated) throughout the experimental period. Air was
constantly aerated through the normoxic tanks to ensure saturation. The
aquaria were maintained at 20°C throughout the acclimation period.

After a 6-week acclimation period, the sustained swimming performance of
each individual fish was tested under both normoxic and hypoxic conditions. In
addition, the mating behaviour of male mosquito fish was measured under both
non-competitive and competitive conditions in both hypoxia and normoxia. For
all experimental tests, the first environment tested for each individual (or
pair) was selected at random. All testing was conducted at 28°C, which
represents a temperature that approximates the optimum for both coercive
mating and swimming performance of male mosquito fish
(Wilson, 2005).

Swimming performance

The sustained swimming performance of each individual male mosquito fish
was tested under normoxic and hypoxic conditions (N=15 for hypoxic
males; N=15 for normoxic males). Sustainable swimming speeds were
measured in a Brett-type swimming flume that consisted of a 10 cm long
swimming section of 3 cm diameter through which a continuous water current was
delivered by an adjustable motor connected to a rotor. Fish were introduced
into the flume at a water velocity of 2 cm s-1 and allowed to
settle for 5 min. The velocity of the current within the flume was then
increased by 2 cm s-1 every 3 min until the fish showed signs of
fatigue. Given the short period between increases in water velocity, the
underlying physiological basis for these measures of swimming performance are
most likely aerobic and anaerobic in nature. A fish was defined as fatigued
when it could no longer hold its position and was swept against the back grid.
The total time to exhaustion and the water velocity at exhaustion were
recorded for each fish and used to calculate the sustained swimming
performance (Umax) with the equation
(Brett, 1964):

where Uf is the highest speed maintained for an entire
3 min interval, Tf is the time taken to exhaustion in the
final speed interval, Ti is the time interval length (3
min), and Ui is the speed increment (2 cm s-1)
[modified from Brett (Brett,
1964)].

Mating behaviour

Mating performance of the males was assessed in a non-competitive and
male-male competitive environment. For the non-competitive tests, an
individual male was given the opportunity to copulate with three mature
females under both hypoxic and normoxic conditions. In all tests of mating
behaviour of male mosquito fish, the test environment referred to both the
partial pressure of oxygen and the state of the female in the specific
environment. Thus, when the mating behaviour of males was tested in hypoxia,
the test environment refers to both low partial pressures of oxygen and
exposure to females acclimated to this hypoxic environment. Differences
between the acclimation groups in each specific environment were still due to
underlying physiological and behavioural changes that were associated with
acclimation. The observation tank consisted of a 30 cm×30 cm×20 cm
deep glass aquarium with aged tapwater (pH 7.0), 2 cm gravel base, a corner
aeration stone and a 200 W Jager heater. Black plastic was placed around the
sides and back of the tank to reduce any external stimuli to the fish. An
aquarium light was also placed above the tank for adequate illumination and to
reduce the ability of the fish to detect movement in the darkened observation
room. Only females that had not given birth during the previous 5 days were
used in the observational experiments because of the possible effects of
post-partum females on male behaviour
(Farr, 1989). All females had a
total body length of between 3.7-3.9 cm. In hypoxic conditions, only females
acclimated to hypoxia were used for mating observations, and vice
versa under normoxia.

Mating behaviour of each male was recorded for a total of 10 min and the
observation time was started when the males first began to follow one of the
females. Mating behaviours were entered into the Behavioural software program
ETHOM 1.0 using a laptop computer. Total time each male spent following the
females (TFT), the total number of mating attempts not including successful
copulations and successful copulations were recorded for each individual
during the observations. Mating attempts were defined as when a male swims
towards a female, swings the gonopodium forward through an angle greater than
90°, and attempts to insert the intromittent organ into the female's
genital opening. Total number of mating attempts included both unsuccessful
mating attempts and successful copulations. Copulations were deemed to occur
when the gonopodium was inserted into the female's genital opening.
Copulations were easily distinguished from unsuccessful mating attempts by the
characteristic twisting motion performed by the male following successful
insertion (Wilson, 2005). This
twisting away from the females is associated with the rapid removal of the
barbed gonopodium from the female's genital opening.

Male mating performance was then tested in a male-male competitive
environment. Competitive experiments involved competing one male from each
acclimation group against each other for the opportunity to copulate with a
single female under both hypoxic and normoxic conditions. As body length is an
important determinant of male territorial and sneaky-mating success in this
species (McPeek, 1992;
Pilastro et al., 1997), males
from each acclimation group were first size-matched with an individual from
the alternate acclimation group. Thus, 15 size-matched pairs of males were
identified and tested. Each pair of size-matched males differed in body length
by less than 0.1 mm. A small portion of the dorsal fin was removed from half
of the males from each treatment so they could be distinguished in the
observation tanks.

Mating behaviour of each competing male was assessed during a 20 min
observation period in a 30 cm×30 cm×20 cm deep aquarium containing
1 mature female mosquito fish. The observation tank was identical to the
set-up for the non-competitive tests. For each pair of fish, a different
female was used for each test environment. To ensure differences in size
between the males and females of each pair were kept constant across the test
environment, each set of females were also size matched. Before observations,
females were introduced into the observation tank and allowed to settle for at
least 20 min. Males were then introduced into the aquarium and the observation
period was started as soon as one male first began to follow the female.

During the 20 min observation period, several male mating behaviours were
recorded and entered into the behavioural software program ETHOM 1.0 using a
laptop computer. TFT, the total number of mating attempts and copulations were
recorded for each individual during the competitive bouts. The total numbers
of dominant behaviours displayed by each male towards the competing male (e.g.
chasing male away, biting male) were also counted for each individual.

Total sperm number per stripped ejaculate was assessed for 17 hypoxic- and
18 normoxic-acclimated males. Ejaculates were stripped from each individual by
palpating their abdomen under a dissecting microscope and diluting the
ejaculate in 2 ml of 0.9% NaCl. A drop of this diluted medium was then placed
on a sperm counting chamber (Hawksley Technology, Lancing, Sussex, UK; depth
10 μm, 0.1×0.1 calibration grid) and the total sperm number was
counted within a known volume of solution (repeated three times with each
sample). Males were separated from females for at least 24 h prior to removal
of ejaculates to ensure sperm stores were unaffected by previous sexual
behaviour (Matthews et al.,
1997).

Statistical analyses

Data were analysed using the statistical programs R and SigmaStat. A
two-way repeated-measures ANOVA was used to test for acute and acclimation
effects on TFT. Total mating attempts, number of copulations and aggressive
behaviours were analysed using R with a generalized linear mixed model (GLM)
using penalized quasi-likelihood using a Poisson distribution to satisfy count
data distributions. TFT was log10 transformed to satisfy normality
assumptions. All data are presented as means ± s.e.m.

For male-male competitive interactions, TFT and ASR were analysed using
one-way ANOVA with pair as a random factor in the statistical program R.
Attempts, copulations and aggressive behaviours were analysed using a one-way
ANOVA with pair as a random factor and a Poisson distribution for count data
in R. Performance trials were analysed using the result of
Umax. A two-way repeated-measures ANOVA was used to
analyse the data in SigmaStat. Sperm counts were analysed using a one-way
ANOVA in R.

Effect of partial pressure of oxygen on the sustained swimming performance
(Umax) of male eastern mosquito fish Gambusia
holbrooki acclimated to either hypoxic or normoxic conditions for at
least 4 weeks. Values are means ± s.e.m., N=15 for each
acclimation group. *Significant results at P<0.05.

Results

We found the effect of partial pressure of oxygen on
Umax differed significantly between treatment groups
(two-way repeated measures ANOVA, F1,60=4.23,
P=0.048; Fig. 1).
Although there was no difference between acclimation groups when tested under
normoxic conditions, the hypoxic-acclimated group had a
Umax that was significantly greater under
hypoxicconditions than normoxic fish (post-hoc test;
P<0.05).

Male mating behaviour in a non-competitive environment was influenced by
long-term exposure to hypoxic conditions
(Fig. 2). The relationship
between partial pressure of oxygen and TFT was significantly influenced by
acclimation treatment (two-way repeated measures ANOVA,
F1,24=5.608, P=0.026). When tested under hypoxic
conditions, hypoxia-acclimated males spent 225.6±18.6 s (N=15)
following females, which was significantly greater than for normoxic-males
(130.0±24.3 s N=15; Fig.
2A). By contrast, there was no significant interaction between
treatment group and test environment for the total number of mating attempts
(GLM, t=1.035, N=26, d.f.=24, P=0.311;
Fig. 2B). No difference between
test environments (GLM, t=-0.13, N=26, d.f.=24,
P=0.90) or treatment groups (GLM, t=-0.18, N=26,
d.f.=24, P=0.86) were detected for number of mating attempts.
However, hypoxic-acclimated males obtained significantly greater number of
copulations than normoxic males (GLM, t=-3.84, N=26,
d.f.=24, P<0.001). Although no differences were detected between
groups when tested under normoxic conditions, hypoxic-acclimated males
(2.8±0.7 copulations N=15) obtained significantly more
copulations in hypoxic conditions than normoxic-acclimated males
(1.5±0.4 copulations N=15;
Fig. 2C).

Male mosquito fish exposed to hypoxia for at least 6 weeks possessed
1.5×105±0.27×105 (N=17)
sperm cells per ejaculate, which was significantly fewer than the
3.9×105±0.83×105 (N=18) for
the normoxic males (P<0.05).

Discussion

The present study represents one of the first experimental investigations
of the benefits of reversible phenotypic plasticity for reproductive
performance. Previous tests of the benefits of acclimation responses have
investigated the fitness consequences of developmental acclimation responses,
with most studies rejecting the generality of the assumption that all
acclimation changes are beneficial (Leroi
et al., 1994; Zamudio et al.,
1995; Huey and Berrigan,
1996; Bennett and Lenski,
1997; Huey et al.,
1999; Wilson and Franklin,
2002). However, most physiological studies of acclimation deal
with reversible responses in adult organisms, and the absence of information
on the ecological significance of these responses represent an important gap
in our understanding of the evolution of phenotypic plasticity. In this study,
we found that both locomotor performance and mating behaviour were
substantially influenced by long-term exposure to low partial pressures of
oxygen. Sustained swimming performance of male mosquito fish was greater in a
hypoxic environment following long-term exposure to low partial pressures of
oxygen. Similarly, in a non-competitive environment, male mosquito fish
acclimated to hypoxic conditions spent a greater amount of time following
females and obtained more copulations than normoxic-acclimated males when
tested in low partial pressures of oxygen.

Effect of partial pressure of oxygen on the mating behaviour
(non-competitive environment) of male eastern mosquito fish Gambusia
holbrooki acclimated to either hypoxic or normoxic conditions for at
least 6 weeks. (A) The total time males spent following females, (B) total
number of attempted copulations and (C) the total number of successful
copulations recorded during a 10 min observation period in a non-competitive
environment. Values are means ± s.e.m., N=15 for each
acclimation group. *Significant results at P<0.05.

Effect of partial pressure of oxygen on the mating behaviour (male-male
competitive environment) of male eastern mosquito fish Gambusia
holbrooki acclimated to either hypoxic or normoxic conditions for at
least 6 weeks. (A) The total time males spent following females, (B) total
number of attempted copulations and (C) the total number of successful
copulations recorded during a 10 min observation period in a male-male
competitive environment. Values are means ± s.e.m., N=15 for
each acclimation group. *Significant results at
P<0.05.

Improvements in the coercive mating ability of male mosquito fish with
acclimation were most likely due to modifications in underlying physiological
traits that were associated with aerobic capacity. Increased sustained
swimming performance and decreased reliance on ASR indicate the greater
aerobic capabilities of male mosquito fish in hypoxic conditions following
acclimation to low partial pressures of oxygen. Previous studies reporting
higher haematocrit, haemoglobin and erythrocyte concentrations of acclimated
to hypoxic conditions (Frey et al.,
1998; Timmerman and Chapman,
2004b; Jensen et al.,
1993), suggest these traits may be the driving underlying
physiological mechanisms. Timmerman and Chapman
(Timmerman and Chapman, 2004b)
also suggest that changes in lower critical oxygen tension and gill surface
area in hypoxic-acclimated fish may support a greater aerobic capacity under
hypoxic conditions.

Despite the advantages of a greater aerobic capacity for hypoxic-acclimated
males in hypoxic environments, benefits to mating performance disappeared when
tested in a competitive environment. We found no influence of long-term
exposure to different partial pressures of oxygen on the mating behaviour of
male mosquito fish in a competitive environment. Thus, it seems the mechanisms
used to compensate for acute hypoxia, such as increasing opercular beat rate
(Randall, 1970) and ASR
(Kramer and Mehegan, 1981),
seem to be able to procure sufficient oxygen to maintain reproductive
behaviours in hypoxia for a short amount of time, before an oxygen debt is
acquired (Vandenthillart and Verbeek,
1991). Given the benefits for the mating behaviour of male
mosquito fish appear to be context dependent, interpreting the ecological
significance of these reversible acclimation responses is difficult
(Woods and Harrison, 2002).
However, when tested in a hypoxic environment, the normoxic-males spent a
greater proportion of time in ASR than hypoxic-acclimated males. Obtaining the
same number of copulations whilst spending more time in ASR, may reflect a
greater energetic cost for maintaining activity for the normoxic-acclimated
males. Longer term analyses of mating behaviour and performance, lasting
several hours, may better reflect the limitations of aerobic capacity on
mating performance.

Effect of partial pressure of oxygen on the time (s) spent in aquatic
surface respiration (ASR; male-male competitive environment) by male eastern
mosquito fish Gambusia holbrooki acclimated to either hypoxic or
normoxic conditions for at least 6 weeks. Values are means ± s.e.m.,
N=15 for each acclimation group. *Significant results at
P<0.05.

Total number of spermatozoa per stripped ejaculate was lower in males that
were exposed to low partial pressures of oxygen. Long-term exposure to hypoxic
conditions could be associated with increased energetic demands, such as
increased costs of maintaining higher haemoglobin, myoglobin and gill filament
surface area, that prevent the production of large quantities of sperm cells.
In addition, prolonged hypoxia may lead to changes in hormone levels that can
retard gonadal development. Hypoxia can produce endocrine disruption in fish
(Wu et al., 2003;
Perry et al., 2004;
Shang and Wu, 2004).
Testosterone, oestrogen and triiodothyronine were reported to decrease in
common carp Cyprinus carpio when acclimated to hypoxia, resulting in
retarded gonadal development (Wu et al.,
2003). Increases in stress hormones such as cortisol as a result
of chronic exposure to hypoxia (Pichavant
et al., 2002) may disrupt spermatogenesis and other hormone
pathways involved in sperm production. Alternatively, it is also possible that
lower sperm numbers could be a physiological response to hypoxic conditions.
Females may not be as reproductively active in these conditions, stimulating a
down-production of sperm in males (Olsen
and Liley, 1993).

Differences in copulation frequency between acclimation groups in the
competitive and non-competitive environments may alternatively be related to
the number of sperm available per ejaculate. In the non-competitive
environment, hypoxia-acclimated males may have obtained more copulations
because they possessed fewer sperm per ejaculate than the normoxia-acclimated
males that may have allocated their sperm more prudently
(Pound and Gage, 2004). By
contrast, in a competitive environment, the lack of difference between the
acclimation groups may be due to a need for all males to maximize copulation
frequency, as predicted by sperm competition theory
(Evans and Magurran, 2001).
However, guppies (P. reticulata) with greater sperm reserves obtained
a greater number of sneaky copulations than males with fewer sperm
(Matthews et al., 1997),
suggesting that normoxia acclimated males should attempt to maximize
copulation frequency across all environments. Also, internally fertilizing
poeciliids show mixed paternity broods and fertilization success is skewed
towards the last male to copulate (Evans
and Magurran, 2001). Thus, sperm number appears to be a poorer
predictor of fertilisation success than the order of copulation
(Evans and Magurran, 2001).
Given this evidence, and the relentless reproductive behaviour of male
mosquito fish, we suggest differences in sperm number and mating success
between the acclimation groups must be a result of physiological performance
in the environment and not male motivation.

Reversible physiological plasticity should be favoured when reliable cues
indicate the state of the environment, and a fitness trade-off occurs between
phenotypes expressed in each of the environments
(Levins, 1968;
Moran, 1992). Despite
substantial improvements in locomotor and mating performance for the
hypoxic-acclimated individuals in a hypoxic environment, there was no obvious
trade-off in a normoxic environment. Some phenotypic modifications to hypoxic
environments may even show short-term benefits in normoxia, supporting
fitness-related aerobic behaviours. In the absence of a fitness trade-off,
theory predicts that the phenotype should be constitutive rather than
inducible (Moran, 1992).
However, long-term costs of these modifications may outweigh short-term
benefits. Long-term exposure to high partial pressures of oxygen was found to
cause acid-base disturbances and an increase in the pH of blood in sea bass
Dicentrarchus labrax (Cecchini and
Caputo, 2003). A similar effect could occur in long-term exposure
to high oxygen in mosquito fish acclimated to hypoxia, negating any benefits
in short-term oxygen procurement. The reduction in sperm production in the
hypoxic-acclimated individuals indicates this may be the case, and studies of
the energetic costs of supporting a modified phenotype in the hypoxic
environment may reveal further costs.

We found the benefits of reversible acclimation responses to the mating
performance of male mosquito fish were dependent on whether they were tested
with or without male-male competition. In hypoxic environments, sustained
aerobic activity and mating performance in the absence of male-male
competition were substantially improved by acclimation to hypoxia. However,
mating performance in a competitive environment was unaffected by long-term
exposure to low partial pressures of oxygen. This represents one of the first
experimental tests of the benefits of reversible acclimation responses, and
suggests the ecological significance of physiological plasticity may be more
complicated than previously thought.

ACKNOWLEDGEMENTS

We thank Stewart Macdonald, Amanda Niehaus and David Putland for
substantial logistical support and the Australian Research Council for
financial assistance.

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